Project Summary Thrombotic cardiovascular diseases are prevalent causes of death and disability in the developed world. Current anti-platelet therapies have not conquered these diseases, and we lack suitable targets for new anti- thrombotic approaches and agents. Platelets are regulated by phosphorylation, but we now find also by remodeling of ubiquitinated proteins. We find platelets continuously ubiquitinate their proteome and continuously remodel these adducted chains. Inhibition of this remodeling by broadly acting deubiquitinase inhibitors suppresses activation in response to any of several agonists within minutes, and blocks platelet adhesion under high shear flow in microfluidic channels. Inhibition of cellular deubiquitinases also sharply suppresses occlusive thrombosis in damaged carotid arteries in vivo. Our data now show that inhibition of specific deubiquitinases suppresses platelet activation. We can inhibit aggregation, adhesion, spreading and migration of washed human platelets by inhibiting the unique deubiquitinase USP7 (Herpes virus-associated ubiquitin specific protease, HAUSP). We also find selective inhibition of just the two proteasome-associated deubiquitinases USP14 and UCHL5, which trim poly-ubiquitin, also inhibited platelet function. This leads us to propose deubiquitinases act in tandem to enable agonist signaling and thrombosis. The essential role of ubiquitin remodeling is also present in megakaryocytes cell lines allowing genetic interventions to confirm our pharmacologic approach. This work shows ubiquitin metabolism regulates platelet reactivity, and shows platelet ubiquitin metabolism can be manipulated. This not only defines new anti-thrombotic targets, our work shows we can successfully intervene in this metabolism in vivo to suppress arterial thrombosis. Moreover, two specific deubiquitinases each have essential roles in platelet action. Specific inhibitors of these enzyme are available and in one case can be safely administered over long times. This allows us to rapidly suppress platelet reactivity through their currently unappreciated roles in thrombosis. Aim 1. Establish the role of targeted deubiquitination in platelet activation and thrombosis. We will identify proteins selectively deubiquitinated by the selective deubiquitinase after stimulation, and will identify proteins in signaling cascades that are regulated by ubiquitin remodeling. Aim 2. Elucidate the role of ubiquitin remodeling in in vivo thrombosis. Here we will determine currently available specific inhibitors suppress intravascular thrombosis.